The determination of the DNA base sequence of the human genome will have a major impact on biomedical science in the next century. The completion of the first complete human DNA will enhance a range of applications from genetic mapping of disease-associated genes to diagnostic tests for disease susceptibility and drug response. The determination of base composition at specific, variable DNA sites known as single nucleotide polymorphisms (SNPs) is especially important. The current generation of sequence determination methods are too slow and costly to meet large-scale SNP analysis requirements. Thus, there is a need for faster, more efficient methods for analyzing genetic sequences for SNPs.
SNPs have a number of uses in mapping, disease gene identification, and diagnostic assays. All of these applications involve the determination of basEs composition at the SNP site. Conventional sequencing can provide this information, but is impractical for screening a large number of sites in a large number of individuals. Several alternative methods have been developed to increase throughput.
Two techniques have been developed to determine base composition at a single site, minisequencing (See, e.g., "Minisequencing: A Specific Tool For DNA Analysis And Diagnostics On Oligonucleotide Arrays," by Tomi Pastinen et al., Genome Research 7, 606 (1997)), and oligo-ligation (See, e.g., "Single-Well Genotyping Of Diallelic Sequence Variations By A Two-Color ELISA-Based Oligonucleotide Ligation Assay," by Vincent O. Tobe et al., Nuclear Acids Res. 24, 3728 (1996)). In minisequencing, a primer is designed to interrogate a specific site on a sample template, and polymerase is used to extend the primer with a labeled dideoxynucleotide. In oligo-ligation, a similar primer is designed, and ligase is used to covalently attach a downstream oligo that is variable at the site of interest. In each case, the preference of an enzyme for correctly base-paired substrates is used to discriminate the base identity that is revealed by the covalent attachment of a label to the primer. In most applications these assays are configured with the primer immobilized on a solid substrate, including microplates, magnetic beads and recently, oligonucleotides microarrayed on microscope slides. Detection strategies include direct labeling with fluorescence detection or indirect labeling using biotin and a labeled streptavidin with fluorescent, chemiluminescent, or absorbance detection.
Oligonucleotide microarrays or "DNA chips" have generated much attention for their potential for massively parallel analysis. The prospect of sequencing tens of thousands of bases of a small sample in just a few minutes is exciting. At present, this technology has limited availability in that arrays to sequence only a handful of genes are currently available, with substantial hardware and consumable costs. In addition, the general approach of sequencing by hybridization is not particularly robust, with the requirement of significant sequence-dependent optimization of hybridization conditions. Nonetheless, the parallelism of an "array" technology is very powerful. and multiplexed sequence determination is an important element of the new flow cytometry method.
Accordingly, it is an object of the present invention to provide a method for determining the base composition at specific sites in a strand of DNA using microspheres and flow cytometry, wherein the specificity of enzymes for discriminating base composition is combined with the parallel analysis of a fluorescent microsphere array.
Additional objects, advantages, and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examinations of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.